Characterization of an Aminopeptidase A from Tetragenococcus halophilus CY54 Isolated from Myeolchi-Jeotgal

In this study, a pepA gene encoding glutamyl (aspartyl)-specific aminopeptidase (PepA; E.C. 3.4.11.7) was cloned from Tetragenococcus halophilus CY54. The translated PepA from T. halophilus CY54 showed very low similarities with PepAs from Lactobacillus and Lactococcus genera. The pepA from T. halophilus CY54 was overexpressed in E. coli BL21(DE3) using pET26b(+). The recombinant PepA was purified by using an Ni– NTA column. The size of the recombinant PepA was 39.13 kDa as determined by SDS-PAGE, while its optimum pH and temperature were pH 5.0 and 60°C, respectively. In addition, the PepA was completely inactivated by 1 mM EDTA, indicating its metallopeptidase nature. The Km and Vmax of the PepA were 0.98 ± 0.006 mM and 0.1 ± 0.002 mM/min, respectively, when Glu-pNA was used as the substrate. This is the first report on PepA from Tetragenococcus species.

94 o C for 30 s, 59 o C for 30 s, 72 o C for 1 min; and final extension at 72 o C for 4 min. The amplified product was ligated with pGEM T-Easy vector (Promega, USA). E. coli DH5α cells were transformed with the ligation mixture by electroporation. Plasmid DNA was prepared from an E. coli transformant (TF) and the insert was sequenced at Cosmogenetech (Korea).

Overexpression of pepA in E. coli and Purification of Recombinant PepA
pepA was amplified as described above and ligated with pET26b(+) (Novagen, USA) (5.36 kb, Kan R). The ligation mixture was used to transform E. coli BL21(DE3) cells by electroporation, and a TF harboring pETA54 (pET26b(+) with pepA) was obtained. LB broth (100 ml) containing kanamycin (60 μg/ml) was inoculated with the TF and the culture was grown at 37 o C until the OD600 reached 0.6. Then, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to 0.5 mM concentration. After 12 h of growth at 20 o C, cells were harvested by centrifugation at 12,000 ×g for 15 min at 4 o C, washed three times with phosphate-buffered saline (PBS, pH 7.4), and resuspended in lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, and 10 mM imidazole, pH 7.0). Cells were then disrupted by sonication (Ultrasonicator, Bandelin Electronic, Germany) and centrifuged at 12,000 ×g for 15 min. The resulting pellet (insoluble fraction) and supernatant (soluble fraction) were examined for PepA by SDS-PAGE. The soluble fraction was loaded onto a Ni-NTA column (GE Healthcare, Sweden). Bound PepA was eluted from the column by stepwise increase in the imidazole concentration (40−500 mM) of the elution buffer. Protein concentration was determined by Bradford method using a BioRad protein assay kit [14]. SDS-PAGE was done using a 10% (w/v) acrylamide gel for separation and a 5% (w/v) gel for stacking.

Enzyme Assay and Properties of Recombinant PepA
The activity of the recombinant PepA was measured by a method previously reported [15], and glu-pNA (Sigma-Aldrich, USA) was used as the substrate. The reaction mixture (500 μl) consisted of 50 μl enzyme solution (10 μg PepA in 50 μl PBS), 400 μl 100 mM potassium phosphate buffer (pH 7.0), and 50 μl 10 mM substrate. The mixture was incubated at 40 o C for 30 min, and then 1 ml 30% acetic acid was added to stop the reaction. The absorbance at 410 nm was measured using a spectrophotometer (UV-1601, Shimadzu, Japan). A standard curve was prepared using various concentrations of para-nitroaniline. One unit of aminopeptidase activity (U) was defined as the amount of enzyme that released 1 nmol of p-nitroaniline in 1 min under the assay conditions.
The effect of pH on the PepA activity was examined. First, purified PepA (10 μg) was incubated for 1 h at 40°C at pH 3-9 and the remaining activities were measured as described above. Buffers of 100 mM concentration were used: citrate-NaOH (pH 3-6), potassium phosphate (pH 6-8), and borate-NaOH (pH [8][9]. The effect of temperature on the PepA activity was then determined after 1 h incubation at 20-90°C (pH 5.0). The effects of NaCl concentration (0-20%), metal ions (2 mM) and protease inhibitors were also determined after 1 h incubation at 60°C and pH 5.0 (100 mM citrate-NaOH buffer). The substrate specificity of PepA was determined with different chromogenic substrates including Glu-pNA, Met-pNA, Ala-pNA, Arg-pNA, Lys-pNA, and Pro-pNA at 60°C and pH 5.0 (100 mM citrate-NaOH buffer). The kinetic parameters of the recombinant PepA were determined at 60°C and pH 5.0 (100mM citrate-NaOH buffer) with Glu-pNA (0.1-2 mM) as the substrate.

Heterologous Expression of pepA in Weissella confusa
pepA was amplified by using the primer pair: F2 (5`-CCCGGATCCTGCTGCTTCATAATG-3`, BamHI site underlined) and R2 (5`-TTTCTGCAGCTGGCAACCTCAATC-3`, PstI site underlined). Amplified pepA was ligated with pHG240E (5.9 kb, EmR) after the BamHI and PstI digestions. pHG240E is a Lactobacillus-E. coli shuttle vector based on a 1.8 kb cryptic plasmid, pHG1, from Levilactobacillus zymae GU240 [16], and an E. coli vector, pBluescriptII KS(+) (2.9 kb, AmpR). The construction of pHG240E will be described elsewhere (manuscript in preparation). The ligation mixture was used to transform E. coli DH5α competent cells. pHG240EpepA (pHG240E with pepA) was prepared from an E. coli transformant and used to transform Weissella confusa CB1 by electroporation. The PepA activities of the TFs were measured as described above.

Cloning of pepA from T. halophilus CY54
T. halophilus CY54 was isolated from myeolchi-jeotgal in 2020 [13]. Among more than 1,000 LAB isolates, T. halophilus CY54 was selected because of its high proteolytic activities on MRS agar plates with skim milk (2%, w/v). T. halophilus CY54 showed desirable properties as a starter for fermented foods with high salinities, which included high proteolytic activity, high salt tolerance, no production of biogenic amines, and sensitivity to medically important antibiotics. The whole genome sequence of T. halophilus CY54 was determined and sequence analyses showed that T. halophilus CY54 possessed 21 putative protease genes and 14 aminopeptidase genes [13]. Among them, a pepA gene encoding glutamyl (aspartyl)-specific aminopeptidase (PepA; E.C. 3.4.11.7) was chosen for further studies. PepA is an exopeptidase which specifically cuts peptides with the N-terminal amino acids aspartic acid and glutamic acid [9]. Peptidases such as PepA contribute to the overall proteolytic capacity of LAB strains and help host cells grow in environments containing proteins such as casein and wheat gluten, both of which are rich in glutamyl/aspartyl [17].

Overexpression of pepA in E. coli BL21(DE3) and Purification of Recombinant PepA
The pepA gene from T. halophilus CY54 was overexpressed in E. coli BL21(DE3) and recombinant PepA was produced in IPTG-induced cells (Fig. 2, lane 2). A control, E. coli BL21(DE3) harboring intact pET26b(+), did not produce PepA (Fig. 2. lane 1). PepA was observed from both soluble and insoluble fractions of cell extract (results not shown). The soluble fraction was used to purify the recombinant PepA since active enzyme was likely present in the soluble fraction rather than in the insoluble fraction. The soluble fraction was loaded onto a Ni-NTA column, and bound PepA was eluted at 100 mM imidazole concentration (Table 1). Sufficiently pure PepA was obtained, and the apparent size was 41 kDa on an SDS gel, matching well with the expected size of PepA with additional His-tag (Fig. 2. lane 3). The size of CY54 PepA is quite similar with those of other characterized LAB PepAs (41 kDa for Lb. delbrueckii subsp. lactis DSM 20072, 39.4 kDa for L. lactis subsp. lactis DSM20481, and 41 kDa for L. lactis subsp. lactis NCDO 712) [17,24].

Properties of Recombinant PepA
The optimal temperature of the recombinant PepA was 60 o C. The activity sharply decreased at 70-80 o C, and no activity was observed at 90 o C (Fig. 3A). The optimum temperature of CY54 PepA was the same as that of Lactobacillus delbrueckii subsp. lactis DSM 20072 (60 o C), lower than that of Lactococcus lactis subsp. lactis NCDO 712 (65 o C), and higher than that of PepA from Streptococcus cremoris HP (50 o C) ( Table 2). The optimum pH for the recombinant PepA was 5.0 (Fig. 3B) Table 2). The effect of metal ions (2 mM) on the PepA activity was examined (Fig. 3C). The  Elution 40 and 100: elution buffer containing 40 and 100 mM imidazole concentration, respectively. activity was increased by CoCl2 (152%), whereas the activity was strongly inhibited by CuSO4 (9%). Other metal ions did not significantly affect the PepA activity. The highest PepA activity was observed at 12% NaCl concentration, which was consistent with the optimum NaCl concentration for the growth of T. halophilus CY54 (Fig. 3D). The effects of various inhibitors and other reagents on the PepA activity were also investigated. SDS, the metallopeptidase inhibitor 1,10-phenanthroline and the metal chelator EDTA strongly inhibited PepA. There was little decrease in the activity by other reagents, indicating that PepA is a metallopeptidase ( Table 3). The substrate specificity of CY54 PepA was examined using 6 sets of pNA substrates. CY54 PepA strongly hydrolyzed Glu-pNA but did not hydrolyze Ala-pNA, Arg-pNA, Lys-pNA, Pro-pNA, and Met-pNA. The kinetic parameters of CY54 PepA were determined using Glu-pNA as the substrate. Km and Vmax were 0.63 ± 0.006 mM and 10.2 ± 0.02 U/mg protein, respectively, while Kcat was 0.0025 ± 0.000021/s, and Kcat/Km was 3.97 ± 0.004/M/s ( Table 2). The Km value of CY54 PepA was higher than those of PepAs from Lc. lactis subsp. lactis DSM 20481 [17] and Lc. lactis subsp. lactis NCDO 712 [24], but lower than that of PepA from Lb. delbrueckii subsp. lactis DSM 20072 [17].  pepA from T. halophilus CY54 was cloned and overexpressed in E. coli BL21 (DE3). The properties of the recombinant PepA were examined. To date, while a few PepAs have been characterized and their properties reported, this is the first report on the characterization of PepA from the genus Tetragenococcus. CY54 PepA is not different significantly from other characterized LAB PepAs in terms of the size, optimum temperature, pH, and inhibition by inhibitors. However, a significant difference is the halophilic nature of CY54 PepA, as it shows the highest activity at 12% NaCl. No data are available for other PepAs. Considering its halophilic nature, CY54 PepA might play important roles in fermenting foods rich in proteins and with high salinities. CY54 PepA can probably be used for the production of flavoring hydrolysates from glutamyl/aspartyl-rich food proteins. Finally, while T. halophilus CY54 can be used as a starter for salted fish and fermented foods with high salt content, further research is needed to better understand the exact role and potential of CY54 PepA.

Conflict of Interest
The authors have no financial conflicts of interests to declare.